Pluripotent human embryonic stem cells (hESCs) can be differentiated in vitro into multiple cell types, including cardiovascular cells (cardiomyocytes, vascular endothelial and smooth muscle cells). The ability to control the growth and differentiation of stem cells in vitro is essential for the successful application of differentiated cells for cell-based therapies. The appropriate cell types committed to a desired lineage, together with the relevant elements of the cardiovascular system, can be used to treat cardiovascular diseases. The proposed research on the identification of the optimal condition for controlling hESC fate uses a novel medium-high throughput microarray platform composed of comprehensive chemical-physical microenvironments. These include (1) immobilized growth factors (GFs) and extracellular matrix proteins (ECMPs), (2) mechanical properties of the ECM, and (3) external mechanical forces acting on the cells. Current knowledge indicates that each of these parameters (i.e., GFs, ECMPs, substrate rigidities, as well as mechanical loadings) plays roles in regulating stem cell fate, but the efficiency is low when acting alone. Since stem cells experience the influence of multiple microenvironmental factors which change during the developmental stages, it is essential to examine the combinatory effects of multi-factorial complexities of the niches, as proposed in the current research. In the proposed research, we will investigate the combinatorial effects of ECM proteins (ECMPs) and GFs on the differentiation of hESCs (Federally approved WA01 and WA09 cell lines). We have designed a hydrogel system to control the rigidities of matrices, covering a range encountered in different tissues, for our ECMP array in stem cell culture. We will also incorporate the flow and stretch devices developed in our lab into the microarray system to assess the roles of external mechanical forces in regulating the cell fate of hESCs on the ECMP/GF/Rigidity platform. This novel combinatory microarray system allows the comprehensive testing of the chemical-physical microenvironment for the choice of optimal conditions for hESC growth and differentiation. The use of such optimally chosen hESCs for translation to cardiovascular tissue engineering will provide the opportunity to significantly advance the development of artificial vessels, angiogenesis patches, as well as cell replacement for heart failure, which will in turn improve the healthcare of patients with cardiovascular diseases and the wellbeing of our citizens. PUBLIC HEALTH RELEVANCE: Pluripotent human embryonic stem cells can be differentiated into multiple cell types, including cells in the cardiovascular systems. The appropriate cell types with the associated networks of vascular cells can be used to treat heart, lung, and blood diseases. This project is aiming at developing a novel systematic approach to understand, define, and ultimately control the process of stem cell differentiation, with the ultimate goal of developing tools of regenerative medicine to treat cardiovascular diseases.